1. Field of the Invention
This invention relates to an analysis method for determining physical density or activity of a specific biochemical component contained in a liquid sample (e.g., blood or urine) dripped on a dry chemical analysis element having a reagent layer by measuring change in optical density of a reacted portion on the dry chemical analysis element where color reaction has occurred, wherein the color reaction is chemical reaction, biochemical reaction or immunoreaction between the reagent layer and the specific biochemical component.
2. Description of the Related Art
Recently, there has been put into practice a dry chemical analysis element of an integrated multi-layered type, which is used to determine physical density or activity of a specific chemical component contained in a liquid sample dripped thereon, or to determine physical density of solid component contained in the liquid sample dripped thereon. Further, analysis elements of similar functions, e.g., filter-paper type elements and applications thereof (including both single layer types and multi-layered types) have also been developed and partly put into practice.
When quantitatively analyzing the chemical components or the like contained in the liquid sample using a dry chemical analysis element, the liquid sample is dripped onto the chemical analysis element (onto a spreading layer when the element is provided with the spreading layer or directly onto the reagent layer when the element is not provided with the spreading layer). The dry chemical analysis element is thereafter held at a constant temperature for a predetermined time in an incubator, so that coloring reaction (pigment-generating reaction or color-changing reaction of the reagent) is promoted on the dry chemical analysis element. After the coloring reaction, change in the optical density of the reacted portion is optically measured. That is, measurement light including a wavelength, which is pre-selected according to the combination of the target chemical component and the reagent contained in the reagent layer, is projected onto the dry chemical analysis element to measure the change in the optical density of the reacted portion on the reagent layer. Then the physical density or the activity of the chemical component is determined based on of the measured optical density referring to a predetermined calibration curve depicting the relationship between the physical density or the activity of the chemical component and the change in the optical density.
The dry chemical analysis element of the integrated multi-layered type generally comprises a substrate of an organic polymer and at least one reagent layer formed on the substrate. Preferably, the dry chemical analysis component additionally comprises a spreading layer superposed on the reagent layer. The dry chemical analysis element of the integrated multi-layered type is generally in the form of a film chip of a predetermined shape such as a square or a rectangle. The film chip may be provided with a frame of an organic polymer for facilitating automated handling of the dry chemical analysis element. Also, there has been proposed an analysis technique of using the film chip by itself without use of the frame.
The optical density of the reacted portion can be measured by extracting light of a certain wavelength from the measurement light emitted by a light source using an interference filter or the like, guiding the light of a certain wavelength through optical fibers or the like, focusing the guided light onto a spot on the dry chemical analysis element using a lens, and measuring reflected light from the spot using a photo-detector mounted in a photometric head. Examples of such a technique for measuring the optical density are disclosed in, for example, Japanese Patent Publication No. 5(1993)-72976 and Japanese Unexamined Patent Publication No. 7(1995)-120477.
However, there has been a problem with the above technique for measuring the optical density that accuracy of measurement may be degraded because of non-uniformity in the amount of the liquid sample dripped on the element or because of difference between an accurate sample-dripped position and a measured position. For this reason, a relatively large amount of the liquid sample has been required to maintain the accuracy of measurement at a reasonable level.
In addition, when the measurement light is focused onto an area having no liquid sample spread thereon or onto a boundary area where only an insufficient amount of the liquid sample has been spread, a large error may be included in the measured optical density because strong reflection may occur on such an area despite little or no liquid sample being spread thereon. Thus, it is preferable to focus the measurement light not onto such an area, but onto an area where a sufficient amount of the liquid sample has been spread.
In this respect, the dry chemical analysis element is usually provided with the spreading layer superposed on the reagent layer, so that the liquid sample dripped substantially onto the center of the element may spread isotropically to provide an area capable of effective color reaction which is sufficiently larger than the beam spot of the measurement light. The measurement light is required to have a beam spot of 4-6 mm in diameter so that a sufficiently large amount of the reflected light is obtained to maintain the accuracy of measurement at a reasonable level. The amount of the liquid sample required to spread beyond such a beam spot is about 10 xcexcl. Even if the required amount of the liquid sample is dripped on the spreading layer, about 50% of the liquid sample may constitute a non-uniform component which makes the reflection amount due to the color reaction non-uniform, instead of spreading uniformly over the entire spreading layer. Influence of such spreading characteristics upon the measuring accuracy may be reduced by accurately regulating the dripping amount of the liquid sample. However, the dripping amount of the liquid sample must be regulated by controlling injection and aspiration of the liquid sample with high accuracy, which is extremely difficult in practice.
In practical implementation, there may be a slight difference between the accurate sample-dripped position (i.e., the position where the most active color reaction occurs) and the measured position. When the difference becomes large, a large error may be included in the measured optical density because the measured optical density may reflect the state at the position with an insufficient amount of the liquid sample where only a low degree of the color reaction has occurred. To avoid such an error, the liquid sample is required to be spread over a relatively large area so that the measured position falls within the covered area. In this respect, a relatively large amount of the liquid sample must be collected, which is burdensome for a weak patient (e.g., a patient in a serious state, an old patient or a child) and which may be impossible for a subject such as a small animal.
Describing in detail referring to figures, shown in FIG. 7A is a sectional view of a dry chemical analysis element 1 in a slide-like form including a film chip 2 held by a frame 3. The frame 3 has a circular aperture at the center thereof. The film chip 2 includes a substrate, at least one reagent layer formed on the substrate, and a spreading layer superposed on the reagent layer. FIG. 7B is a plane view of the dry chemical analysis element 1 of FIG. 7A, provided with a sufficient amount of the liquid sample dripped thereon. FIG. 7C is another plane view of the same dry chemical analysis element 1, but provided with only an insufficient amount of the liquid sample dripped thereon. In FIGS. 7B and 7C, the cross-hatched portions P1 and P2 indicate the reacted portions where the color reaction has occurred.
FIG. 8 is a schematic view showing a possible structure of an existing photometric head 50 for measuring the optical density of the reacted portion on the dry chemical analysis element 1. The photometric head 50 in FIG. 8 includes an optical fiber 51 for guiding the measurement light of a suitable wavelength onto a measuring surface of the film chip 2 so that the right angle of incidence can be attained, a collective lens 52 for collecting the light emitted from the optical fiber 51, and a pair of photo-detectors 53 for detecting the light reflected by the measuring surface of the film chip 2. The measurement light from the photometric head 50 is focused onto the reacted portion as a beam having a beam spot 54 of a predetermined radius. The optical density is calculated from the intensity of the reflected light detected by the photo-detectors 53. Then, the physical density or the activity of the target chemical component is determined referring to a predetermined calibration curve representing the relationship between the physical density or the activity of the chemical component and the change in the optical density.
Shown in FIG. 9 are distribution curves for degree of the color reaction on the dry chemical analysis element 1 and the sensitivity of the photometric head 50. Herein, the term xe2x80x9cdegree of the color reactionxe2x80x9d means the same as the term xe2x80x9cthe change in the optical density.xe2x80x9d The curve C1 represents the distribution of the degree of the color reaction for the reacted portion P1 in FIG. 7B on the dry chemical analysis element 1 provided with a sufficient amount of the liquid sample. The curve C2 represents the distribution of the degree of the color reaction for the reacted portion P2 in FIG. 7C on the dry chemical analysis element 1 provided with an insufficient amount of the liquid sample. Each curve indicates that substantially constant degree of the color reaction is attained in the central area, and that the degree of the color reaction gradually decreases in the boundary area. The curve R represents the distribution of the sensitivity of the photometric head 50. The curve R indicates that the sensitivity is high at the center of the beam spot 54 but sharply decreases near the boundary of the beam spot 54.
When projecting the measurement light onto the dry chemical analysis element 1 on which sufficient degree of the color reaction has occurred over the area larger than the beam spot 54, as indicated by the curve C1, an effective result can be obtained as the intensity of the reflected light detected by the photo-detectors 53 accurately reflects the degree of the color reaction throughout the measured area. On the other hand, the result is not effective when the measurement light is projected onto the dry chemical analysis element 1 on which sufficient degree of the color reaction has occurred only within an area smaller than the beam spot 54, as indicated by the curve C2, as the reflected light detected by the photo-detectors 53 includes a light component reflected by the boundary area and/or the area outside the boundary area where only insufficient degree of the color reaction or no color reaction has occurred. The boundary area and/or the area outside the boundary area may reflect the light projected thereon by a larger reflectance than the area where the sufficient color reaction has occurred. Because of such an intense reflected light component which does not reflect the degree of the color reaction, a large error may be included in the calculated degree of the color reaction, making an effective quantitative analysis impossible. Thus, the liquid sample is always required to be spread over a relatively large area to realize the distribution of the degree of the color reaction as indicated by the curve C1. Accordingly, a relatively large amount of the liquid sample must be dripped on the dry chemical analysis element 1. In addition, the sample-dripped position must be accurately controlled to avoid a large separation between the sample-dripped position and the actually measured position.
FIG. 10 is a diagram showing the relationship between the dripped amount of the liquid sample and the degree of the color reaction. As the dripped amount of the liquid sample increases, the area over which the liquid sample is spread becomes larger. Concurrently, the amount of the liquid sample per unit area also increases at each point within the area over which the liquid sample has already been spread, resulting in a higher degree of the color reaction at each point. That is, even if the liquid sample is spread over an area sufficiently larger than the spot 54, the measured degree D of the color reaction may include an error d due to the difference between the dripped amount of the liquid sample, b-a. To minimize the error d, the liquid sample must be dripped accurately by a constant amount.
An object of the present invention is to provide an analysis method using a dry chemical analysis element with which the change in the optical density indicating the composition of a liquid sample can be measured accurately without accurate control of the amount and position of the liquid sample dripped on the dry chemical analysis element.
According to a first aspect of the present invention, there is an analysis method using a dry chemical analysis element comprising the steps of: dripping a liquid sample onto the dry chemical analysis element including a reagent layer, measuring change in optical density of a reacted portion on the dry chemical analysis element where color reaction between the liquid sample and the reagent layer has occurred, and determining physical density or activity of a specific biochemical component contained in the liquid sample; wherein the step of measuring the change in the optical density comprises the steps of: measuring one-dimensional distribution of the change in the optical density along a straight line crossing a central portion of the reacted portion by causing a one-dimensional optical reading apparatus to scan the straight line, and measuring a length of the reacted portion along the straight line crossing the central portion of the reacted portion using the one-dimensional optical reading apparatus; and wherein the physical density or the activity is determined based on an integrated value of the change in the optical density and the measured length of the reacted portion.
According to a second aspect of the present invention, there is an analysis method using a dry chemical analysis element comprising the steps of: dripping a liquid sample onto the dry chemical analysis element including a reagent layer, measuring change in optical density of a reacted portion on the dry chemical analysis element where color reaction between the liquid sample and the reagent layer has occurred, and determining physical density or activity of a specific biochemical component contained in the liquid sample; wherein the step of measuring the change in the optical density comprises the steps of: measuring one-dimensional distribution of the change in the optical density along a straight line crossing a central portion of the reacted portion by causing a one-dimensional optical reading apparatus to scan the straight line, calculating two boundary positions of the reacted portion based on slopes of the obtained one-dimensional distribution of the change in the optical density, and defining distance between the two boundary positions as a length of the reacted portion; and wherein the physical density or the activity is determined based on an integrated value of the change in the optical density and the defined length of the reacted portion.
According to a third aspect of the present invention, there is an analysis method using a dry chemical analysis element comprising the steps of: dripping a liquid sample onto the dry chemical analysis element including a reagent layer, measuring change in optical density of a reacted portion on the dry chemical analysis element where color reaction between the liquid sample and the reagent layer has occurred, and determining physical density or activity of a specific biochemical component contained in the liquid sample; wherein the step of measuring the change in the optical density comprises the steps of: measuring two-dimensional distribution of the change in the optical density over an entire spread area of the reacted portion by causing a two-dimensional optical reading apparatus to scan the entire spread area, and measuring the spread area of the reacted portion using the two-dimensional optical reading apparatus; and wherein the physical density or the activity is determined based on an integrated value of the change in the optical density and the measured spread area.
According to a fourth aspect of the present invention, there is an analysis method using a dry chemical analysis element comprising the steps of: dripping a liquid sample onto the dry chemical analysis element including a reagent layer, measuring change in optical density of a reacted portion on the dry chemical analysis element where color reaction between the liquid sample and the reagent layer has occurred, and determining physical density or activity of a specific biochemical component contained in the liquid sample; wherein the step of measuring the change in the optical density comprises the steps of: measuring two-dimensional distribution of the change in the optical density over an entire spread area of the reacted portion by causing a two-dimensional optical reading apparatus to scan the entire spread area, calculating a boundary of the reacted portion based on slopes of the measured two-dimensional distribution of the change in the optical density, and defining an area within the calculated boundary as the spread area of the reacted portion; and wherein the physical density or the activity is determined based on an integrated value of the change in the optical density and the measured spread areaof the reacted portion.
According to the above analysis methods of the present invention, the change in the optical density can be measured with a stable accuracy even if the center of the straight line or the spread area scanned by the one-dimensional or two-dimensional optical reading apparatus does not match accurately with the exact center of the reacted portion. Thus, sufficiently accurate measurement can be carried out regardless of the fluctuation of the amount and/or position of the liquid sample dripped on the dry chemical analysis element. In addition, the measurement can be carried out with a sufficient accuracy requiring only a small amount of the liquid sample. Accordingly, the entire process of analysis is simplified to provide an easy analysis method at a low cost.